Porous particles comprising excipients for deep lung delivery

ABSTRACT

Improved porous particles for drug delivery to the pulmonary system, and methods for their synthesis and administration are provided. In a preferred embodiment, the porous particles are made of a biodegradable material and have a mass density less than 0.4 g/cm 3 /. The particles may be formed of biodegradable materials such as biodegradable polymers. For example, the particles may be formed of a functionalized polyester graft copolymer consisting of a linear a-hydroxy-acid polyester backbone having at least one amino acid group incorporated therein and at least one poly(amino acid) side chain extending from an amino acid group in the polyester backbone. In one embodiment, porous particles having a relatively large mean diameter, for example greater than 5 μm, can be used for enhanced delivery of a therapeutic agent to the alveolar region of the lung. The porous particles incorporating a therapeutic agent may be effectively aerosolized for administration to the respiratory tract to permit systemic or local delivery of wide variety of therapeutic agents.

RELATED APPLICATIONS

[0001] This application is a Continuation of U.S application Ser. No.10/209,654, filed on Jul. 30, 2002, which is a Continuation of U.S.application Ser. No. 09/888,688 filed on Jun. 25, 2001 which is aContinuation of U.S. application Ser. No. 09/569,153 filed on May 11,2000, now U.S. Pat. No. 6,254,854, which is a Continuation of U.S.application Ser. No. 08/655,570, filed May 24, 1996, now abandoned; theentire teachings of all referenced applications and patents areincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to porous polymericparticles for drug delivery to the pulmonary system.

[0003] Biodegradable polymeric particles have been developed for thecontrolled-release and delivery of protein and peptides drugs. Langer,R., Science, 249: 527-1533 (1990). Examples include the use ofbiodegradable particles for gene therapy (Mulligan, R. C., Science,260:926-932 (1993)) and “single-shot” vaccine delivery (Eldridge et al.,Mol. Immunol., 28:287-294 (1991)) for immunization. Protein and peptidedelivery via degradable particles is restricted due to lowbioavailability in the blood stream, since macromolecules and/ormicroparticles tend to poorly permeate organ-blood barriers of the humanbody, particularly when delivered either orally or invasively.

[0004] Aerosols for the delivery of therapeutic agents to therespiratory tract have been developed. The respiratory tract includesthe upper airways, including the oropharynx

[0005] Aerosols for the delivery of therapeutic agents to therespiratory tract have been developed. The respiratory tract includesthe upper airways, including the oropharynx and larynx, followed by thelower airways, which include the trachea followed by bifurcations intothe bronchi and bronchioli. The upper and lower airways are called theconductive airways. The terminal bronchioli then divide into respiratorybronchioli which then lead to the ultimate respiratory zone, thealveoli, or deep lung. Gonda, I. “Aerosols for delivery of therapeuticand diagnostic agents to the respiratory tract,” In Critical Reviews inTherapeutic Drug Carrier Systems, 6:273-313, (1990). The deep lung, oralveoli, are the primary target of inhaled therapeutic aerosols forsystemic delivery.

[0006] Inhaled aerosols have been used for the treatment of local lungdisorders including asthma and cystic fibrosis (Anderson, et al., Am.Rev. Rev. Respir. Dis., 140:1317-1324 (1989)) and have potential for thesystemic delivery of peptides and proteins as well (Patton and Platz,Advanced Drug Delivery Reviews, 8:179-196 (1992)). However, pulmonarydrug delivery strategies present many difficulties for the delivery ofmacromolecules; these include protein denaturation duringaerosolization, excessive loss of inhaled drug in the oropharyngealcavity (typically exceeding 80%), poor control over the site ofdeposition, irreproducibility of therapeutic results owing to variationsin breathing patterns, the quick absorption of drug potentiallyresulting in local toxic effects, and phagocytosis by lung macrophages.

[0007] Local and systemic inhalation therapies can often benefit from arelative slow controlled release of the therapeutic agent. Gonda, I.,“Physico-chemical principles in aerosol delivery,” In Topics inPharmaceutical Science, 1991, D. J. A. Crommelin and K. K. Midha, Eds.,Stuttgart: Medpharm Scientific Publishers, pp. 95-117, 1992. Slowrelease from a therapeutic aerosol can prolong the residence of anadministered drug in the airways or acini, and diminish the rate of drugappearance in the blood stream. Also, patient compliance is increased byreducing the frequency of dosing. Langer, R., Science, 249:1527-1533(1990); and Gonda, I., “Aerosols for delivery of therapeutic anddiagnostic agents to the respiratory tract,” In Critical Reviews inTherapeutic Drug Carrier Systems, 6:273-313, 1990.

[0008] The human lungs can remove or rapidly degrade hydrolyticallycleavable deposited aerosols over periods ranging from minutes to hours.In the upper airways, ciliated epithelia contribute to the “mucociliaryexcalator” by which particles are swept from the airways toward themouth. Pavia, D., “LungMucociliary Clearance,” In Aerosols and the Lung:Clinical and Experimental Aspects, Clarke, S. W. and Pavia, D., Eds.,Butterworths, London, 1984. In the deep lungs, alveolar macrophages arecapable of phagocytosing particles soon after their deposition. Warheit,M. B. and Hartsky, M. A., Microscopy Res. Tech., 26:412-422 (1993); andBrain, J. D., “Physiology and Pathophysiology of Pulmonary Macrophages,”In The Reticuloendothelial System, S. M. Reichard and J. Filkins, Eds.,Plenum, New York, pp. 315-327, 1985. As the diameter of particlesexceeds 3 μm, there is increasingly less phagocytosis by macrophages.However, increasing the particle size also minimizes the probability ofparticles (possessing standard mass density) entering the airways andacini due to excessive deposition in the oropharyngeal or nasal regions.Heyder, J., et al., J. Aerosol Sci., 17: 811-825 (1986). An effectiveslow-release inhalation therapy requires a means of avoiding orsuspending the lung's natural clearance mechanisms until drugs have beeneffectively delivered.

[0009] Therapeutic dry-powder aerosols have been made as solid(macroscopically nonporous) particles, with mean diameters less thanapproximately 5 μm to avoid excessive oropharyngeal deposition.Ganderton, D., J. Biopharmaceutical Sciences, 3:101-105 (1992); andGonda, I., “Physico-Chemical Principles in Aerosol Delivery,” In Topicsin Pharmaceutical Sciences, 1991, Commelin, D. J. and K. K. Midha, Eds.,Medpharm Scientific Publishers, Stuttgart, pp. 95-115, 1992.

[0010] There is a need for improved inhaled aerosols for pulmonarydelivery of therapeutic agents. There is a need for the development ofdrug carriers which are capable of delivering the drug in an effectiveamount into the airways or the alveolar zone of the lung. There furtheris a need for the development of drug carriers for use as inhaledaerosols which are biodegradable and are capable of controlled releasewithin the airways or in the alveolar zone of the lung.

[0011] It is therefore an object of the present invention to provideimproved carriers for the pulmonary delivery of therapeutic agents. Itis a further object of the invention to provide inhaled aerosols whichare effective carriers for delivery of therapeutic agents to the deeplung. It is another object of the invention to provide carriers forpulmonary delivery which avoid phagocytosis in the deep lung. It is afurther object of the invention to provide carriers for pulmonary drugdelivery which are capable of biodegrading and releasing the drug at acontrolled rate.

SUMMARY OF THE INVENTION

[0012] Improved porous particles for drug delivery to the pulmonarysystem, and methods for their synthesis and administration are provided.In a preferred embodiment, the porous particles are made of abiodegradable material and have a mass density less than 0.4 g/cm³. Theparticles may be formed of biodegradable materials such as biodegradablepolymers. For example, the particles may be formed of a functionalizedpolyester graft copolymer consisting of a linear .alpha.-hydroxy-acidpolyester backbone having at least one amino acid group incorporatedtherein and at least one poly(amino acid) side chain extending from anamino acid group in the polyester backbone. In one embodiment, porousparticles having a relatively large mean diameter, for example, greaterthan 5 μm, can be used for enhanced delivery of a therapeutic agent tothe airways or the alveolar region of the lung. The porous particlesincorporating a therapeutic agent may be effectively aerosolized foradministration to the respiratory tract to permit systemic or localdelivery of a wide variety of therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a graph comparing total particle mass of porous andnon-porous particles deposited on the nonrespirable and respirablestages of a cascade impactor following aerosolization.

[0014]FIG. 2 is a graph comparing total particle mass deposited in thetrachea and after the carina (lungs) in rat lungs and upper airwaysfollowing intratracheal aerosolization during forced ventilation ofporous PLAL-Lys particles and non-porous PLAL particles.

[0015]FIG. 3 is a graph comparing total particle recovery of porousPLAL-Lys particles and non-porous PLAL particles in rat lungs andfollowing broncho alveolar lavage.

DETAILED DESCRIPTION OF THE INVENTION

[0016] Biodegradable particles for improved delivery of therapeuticagents to the respiratory tract are provided. The particles can be usedin one embodiment for controlled systemic or local drug delivery to therespiratory tract via aerosolization. In one embodiment, the particlesare porous particles having a mass density less than 1.0 g/cm³,preferably less than about 0.4 g/cm³. The porous structure permits deeplung delivery of relatively large diameter therapeutic aerosols, forexample greater than 5 μm in mean diameter. The particles also mayinclude a rough surface texture which can reduce particle agglomerationand provide a highly flowable powder, which is ideal for aerosolizationvia dry powder inhaler devices, leading to lower deposition in the mouthand throat.

[0017] Mass Density and Diameter of Porous Particles

[0018] As used herein the term “porous particles” refers to particleshaving a total mass density less than about 0.4 g/cm³. The mean diameterof the particles can range, for example, from about 100 nm to 15 μm, orlarger depending on factors such as particle composition, and thetargeted site of the respiratory tract for deposition of the particle.

[0019] Particle Size

[0020] In one embodiment, particles which are macroscopically porous,and incorporate a therapeutic drug, and having a mass density less thanabout 0.4 g/cm³, can be made with mean diameters greater than 5 μm, suchthat they are capable of escaping inertial and gravitational depositionin the oropharyngeal region, and are targeted to the airways or the deeplung. The use of larger porous particles is advantageous since they areable to aerosolize more efficiently than smaller, non-porous aerosolssuch as those currently used for inhalation therapies.

[0021] The large (>5 μm) porous particles are also advantageous in thatthey can more successfully avoid phagocytic engulfment by alveolarmacrophages and clearance from the lungs, in comparison to smallernon-porous particles, due to size exclusion of the particles from thephagocytes' cytosolic space. Phagocytosis of particles by alveolarmacrophages diminishes precipitously as particle diameter increasesbeyond 3 μm. Kawaguchi, H. et al., Biomaterials, 7:61-66 (1986); Krenis,L. J. and Strauss, B., Proc. Soc. Exp. Med., 107:748-750 (1961); andRudt, S. and Muller, R. H., J. Contr. Rel., 22:263-272 (1992). Theporous particles thus are capable of a longer term release of atherapeutic agent. Following inhalation, porous degradable particles candeposit in the lungs (due to their relatively low mass density), andsubsequently undergo slow degradation and drug release, without themajority of the particles being phagocytosed by alveolar macrophages.The drug can be delivered relatively slowly into the alveolar fluid, andat a controlled rate into the blood stream, minimizing possible toxicresponses of exposed cells to an excessively high concentration of thedrug. The porous polymeric particles thus are highly suitable forinhalation therapies, particularly in controlled release applications.The preferred diameter for porous particles for inhalation therapy isgreater than 5 μm, for example between about 5-15 μm.

[0022] The particles also may be fashioned with the appropriatematerial, diameter and mass density for localized delivery to otherregions of the respiratory tract such as the upper airways. For examplehigher density or larger particles may be used for upper airwaydelivery.

[0023] Particle Deposition

[0024] Inertial impaction and gravitational settling of aerosols arepredominant deposition mechanisms in the airways and acini of the lungsduring normal breathing conditions. Edwards, D. A., J. Aerosol Sci.,26:293-317 (1995). Both deposition mechanisms increase in proportion tothe mass of aerosols and not to particle volume. Since the site ofaerosol deposition in the lungs is determined by the intrinsic mass ofthe aerosol (at least for particles of mean aerodynamic diameter greaterthan approximately 1 μm), diminishing particle mass density byincreasing particle porosity permits the delivery of larger particlesinto the lungs, all other physical parameters being equal.

[0025] The low mass porous particles have a small aerodynamic diameterin comparison to the actual sphere diameter. The aerodynamic diameter,d_(ae), is related to the actual sphere diameter, d (Gonda, I.,“Physico-chemical principles in aerosol delivery,” In Topics inPharmaceutical Sciences, 1991, (eds. D. J. A. Crommelin and K. K.Midha), pp. 95-117, Stuttgart: Medpharm Scientific Publishers, 1992) bythe formula:

d_(aer)=d_({square root}ρ)

[0026] where the particle mass density ρ is in units of g/cm³. Maximaldeposition of monodisperse aerosol particles in the alveolar region ofthe human lung (˜60%) occurs for an aerodynamic diameter ofapproximately d_(aer)=3 μm. Heyder, J. et al., J. Aerosol Sci., 17:811-825 (1986). Due to their small mass density, the actual diameter dof porous particles comprising a mondisperse inhaled powder that willexhibit maximum deep-lung deposition is:

d=3/{square root}ρμm (where ρ<1);

[0027] where d is always greater than 3 μm. For example, porousparticles that display a mass density, ρ=0.1 g/cm³, will exhibit amaximum deposition for particles having actual diameters as large as 9.5μm. The increased particle size diminishes interparticle adhesionforces. Visser, J., Powder Technology, 58:1-10. Thus, large particlesize increases efficiency of aerosolization to the deep lung forparticles of low mass density.

[0028] Particle Materials

[0029] The porous particles preferably arc biodegradable andbiocompatible, and optionally are capable of biodegrading at acontrolled rate for delivery of a drug. The porous particles can be madeof any material which is capable of forming a porous particle having amass density less than about 0.4 g/cm³. Both inorganic and organicmaterials can be used. For example, ceramics may be used. Othernon-polymeric materials may be used which are capable of forming porousparticles as defined herein.

[0030] Polymeric Particles

[0031] The particles may be formed from any biocompatible, andpreferably biodegradable polymer, copolymer, or blend, which is capableof forming porous particles having a density less than about 0.4 g/cm³.

[0032] Surface eroding polymers such as polyanhydrides may be used toform the porous particles. For example, polyanhydrides such aspoly[(p-carboxyphenoxy)hexane anhydride] (“PCPH”) may be used.Biodegradable polyanhydrides are described, for example, in U.S. Pat.No. 4,857,311, the disclosure of which is incorporated herein byreference.

[0033] In another embodiment, bulk eroding polymers such as those basedon polyesters including poly(hydroxy acids) can be used. For example,polyglycolic acid (“PGA”) or polylactic acid (“PLA”) or copolymersthereof may be used to form the porous particles, wherein the polyesterhas incorporated therein a charged or functionalizable group such as anamino acid as described below.

[0034] Other polymers include polyamides, polycarbonates, polyalkylenessuch as polyethylene, polypropylene, poly(ethylene glycol),poly(ethylene oxide), poly(ethylene terephthalate), polyvinyl compoundssuch as polyvinyl alcohols, polyvinyl ethers, and polyvinyl esters,polymers of acrylic and methacrylic acids, celluloses, polysaccharides,and peptides or proteins, or copolymers or blends thereof which arecapable of forming porous particles with a mass density less than about0.4 g/cm³. Polymers may be selected with or modified to have theappropriate stability and degradation rates in vivo for differentcontrolled drug delivery applications.

[0035] Polyester Graft Copolymers

[0036] In one preferred embodiment, the porous particles are formed fromfunctionalized polyester graft copolymers, as described in Hrkach etal., Macromolecules, 28:4736-4739 (1995); and Hrkach et al.,“Poly(L-Lactic acid-co-amino acid) Graft Copolymers: A Class ofFunctional Biodegradable Biomaterials” In Hydrogel and BiodegradablePolymers for Bioapplications, ACS Symposium Series No. 627, Raphael M.Ottenbrite et al., Eds., American Chemical Society, Chapter 8, pp.93-101, 1996, the disclosures of which are incorporated herein byreference. The functionalized graft copolymers are copolymers ofpolyesters, such as poly(glycolic acid) or poly(lactic acid), andanother polymer including functionalizable or ionizable groups, such asa poly(amino acid). In a preferred embodiment, comb-like graftcopolymers are used which include a linear polyester backbone havingamino acids incorporated therein, and poly(amino acid) side chains whichextend from the amino acid groups in the polyester backbone. Thepolyesters may be polymers of α-hydroxy acids such as lactic acid,glycolic acid, hydroxybutyric acid and valeric acid, or derivatives orcombinations thereof. The inclusion of ionizable side chains, such aspolylysine, in the polymer has been found to enable the formation ofmore highly porous particles, using techniques for making microparticlesknown in the art, such as solvent evaporation. Other ionizable groups,such as amino or carboxyl groups, may be incorporated, covalently ornoncovalently, into the polymer to enhance porosity. For example,polyaniline could be incorporated into the polymer.

[0037] An exemplary polyester graft copolymer, which may be used to formporous polymeric particles is the graft copolymer, poly(lacticacid-co-lysine-graft-lysine) (“PLAL-Lys”), which has a polyesterbackbone consisting of poly(L-lactic acid-co-Z-L-lysine) (PLAL), andgrafted lysine chains. PLAL-Lys is a comb-like graft copolymer having abackbone composition, for example, of 98 mol % lactic acid and 2 mol %lysine and poly(lysine) side chains extending from the lysine sites ofthe backbone.

[0038] PLAL-Lys may be synthesized as follows. First, the PLAL copolymerconsisting of L-lactic acid units and approximately 1-2%Nε-carbobenzoxy-L-lysine (Z-L-lysine) units is synthesized as describedin Barrera et al., J. Am. Chem. Soc., 115:11010 (1993). Removal of the Zprotecting groups of the randomly incorporated lysine groups in thepolymer chain of PLAL yields the free e-amine which can undergo furtherchemical modification. The use of the poly(lactic acid) copolymer isadvantageous since it biodegrades into lactic acid and lysine, which canbe processed by the body. The existing backbone lysine groups are usedas initiating sites for the growth of poly(amino acid) side chains.

[0039] The lysine ε-amine groups of linear poly(L-lacticacid-co-L-lysine) copolymers initiate the ring opening polymerization ofan amino acid N-carboxyanhydride (NCA) to produce poly(L-lacticacid-co-amino acid) comb-like graft copolymers. In a preferredembodiment, NCAs are synthesized by reacting the appropriate amino acidwith triphosgene. Daly et al., Tetrahedron Lett., 29:5859 (1988). Theadvantage of using triphosgene over phosgene gas is that it is a solidmaterial, and therefore, safer and easier to handle. It also is solublein THF and hexane so any excess is efficiently separated from the NCAs.

[0040] The ring opening polymerization of amino acid N-carboxyanhydrides(NCAs) is initiated by nucleophilic initiators such as amines, alcohols,and water. The primary amino initiated ring opening polymerization ofNCAs allows good control over the degree of polymerization when themonomer to initiator ratio (M/I) is less than 150. Kricheldorf, H. R. InModels of Biopolymers by Ring-Opening Polymerization, Penezek, S., Ed.,CRC Press, Boca Raton, 1990, Chapter 1; Kricheldorf, H. R.,α-Aminoacid-N-Carboxy-Anhydrides and Related Heterocycles,Springer-Verlag, Berlin, 1987; and Imanishi, Y. In Ring-OpeningPolymerization, Ivin, K. J. and Saegusa, T., Eds., Elsevier, London,1984, Volume 2, Chapter 8. Methods for using lysine ε-amine groups aspolymeric initiators for NCA polymerizations are described in the art.Sela, M. et al., J. Am. Chem. Soc., 78:746 (1956).

[0041] In the reaction of an amino acid NCA with PLAL, the nucleophilicprimary ε-amine of the lysine side chain attacks C-5 of the NCA leadingto ring opening and formation of the amino acid amide along with theevolution of CO₂. Propagation takes place via further attack of theamine group of the amino acid amides on subsequent NCA molecules. Thedegree of polymerization of the poly(amino acid) side chains, thecorresponding amino acid content in the graft copolymers and theirresulting physical and chemical characteristics can be controlled bychanging the M/I ratio for the NCA polymerization—that is, changing theratio of NCA to lysine ε-amine groups of pLAL. Thus, in the synthesis,the length of the poly(amino acid), such as poly(lysine), side chainsand the total amino acid content in the polymer may be designed andsynthesized for a particular application.

[0042] The poly(amino acid) side chains grafted onto or incorporatedinto the polyester backbone can include any amino acid, such as asparticacid, alanine or lysine, or mixtures thereof. The functional groupspresent in the amino acid side chains, which can be chemically modified,include amino, carboxylic acid, sulfide, guanidino, imidazole andhydroxyl groups. As used herein, the term “amino acid” includes naturaland synthetic amino acids and derivatives thereof. The polymers can beprepared with a range of amino acid side chain lengths, for example,about 10-100 or more amino acids, and with an overall amino acid contentof, for example, 7-72% or more depending on the reaction conditions. Thegrafting of poly(amino acids) from the pLAL backbone may be conducted ina solvent such as dioxane, DMF, or CH₂Cl₂ or mixtures thereof. In apreferred embodiment, the reaction is conducted at room temperature forabout 2-4 days in dioxane.

[0043] Alternatively, the porous particles for pulmonary drug deliverymay be formed from polymers or blends of polymers with differentpolyester/amino acid backbones and grafted amino acid side chains. Forexample, poly(lactic acid-co-lysine-graft-alanine-lysine)(“PLAL-Ala-Lys”), or a blend of PLAL-Lys with poly(lacticacid-co-glycolic acid-block-ethylene oxide) (“PLGA-PEG”)(“PLAL-Lys-PLGA-PEG”) may be used.

[0044] In the synthesis, the graft copolymers may be tailored tooptimize different characteristic of the porous particle including: i)interactions between the agent to be delivered and the copolymer toprovide stabilization of the agent and retention of activity upondelivery; ii) rate of polymer degradation and, thereby, rate of drugrelease profiles; iii) surface characteristics and targetingcapabilities via chemical modification; and iv) particle porosity.

[0045] Formation of Porous Polymeric Particles

[0046] Porous polymeric particles may be prepared using single anddouble emulsion solvent evaporation, spray drying, solvent extractionand other methods well known to those of ordinary skill in the art.Methods developed for making microspheres for drug delivery aredescribed in the literature, for example, as described by Mathiowitz andLanger, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al.,Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl.Polymer Sci. 35:755-774 (1988), the teachings of which are incorporatedherein. The selection of the method depends on the polymer selection,the size, external morphology, and crystallinity that is desired, asdescribed, for example, by Mathiowitz, et al., Scanning Microscopy,4:329-340 (1990); Mathiowitz, et al., J. Appl. Polymer Sci., 45:125-134(1992); and Benita, et al., J. Pharm. Sci., 73:1721-1724 (1984), theteachings of which are incorporated herein.

[0047] In solvent evaporation, described for example, in Mathiowitz, etal., (1990), Benita, and U.S. Pat. No. 4,272,398 to Jaffe, the polymeris dissolved in a volatile organic solvent, such as methylene chloride.Several different polymer concentrations can be used, for example,between 0.05 and 0.20 g/ml. The drug, either in soluble form ordispersed as fine particles, is added to the polymer solution, and themixture is suspended in an aqueous phase that contains a surface activeagent such as poly(vinyl alcohol). The aqueous phase may be, forexample, a concentration of 1% poly (vinyl alcohol) w/v in distilledwater. The resulting emulsion is stirred until most of the organicsolvent evaporates, leaving solid microspheres, which may be washed withwater and dried overnight in a lyophilizer.

[0048] Microspheres with different sizes (1-1000 microns) andmorphologies can obtained by this method which is useful for relativelystable polymers such as polyesters and polystryrene. However, labilepolymers such as polyanhydrides may degrade due to exposure to water.For these polymers, solvent removal may be preferred.

[0049] Solvent removal was primarily designed for use withpolyanhydrides. In this method, the drug is dispersed or dissolved in asolution of a selected polymer in a volatile organic solvent likemethylene chloride. The mixture is then suspended in oil, such assilicon oil, by stirring, to form an emulsion. Within 24 hours, thesolvent diffuses into the oil phase and the emulsion droplets hardeninto solid polymer microspheres. Unlike solvent evaporation, this methodcan be used to make microspheres from polymers with high melting pointsand a wide range of molecular weights. Microspheres having a diameterfor example between one and 300 microns can be obtained with thisprocedure.

[0050] Targeting of Particles

[0051] Targeting molecules can be attached to the porous particles viareactive functional groups on the particles. For example, targetingmolecules can be attached to the amino acid groups of functionalizedpolyester graft copolymer particles, such as PLAL-Lys particles.Targeting molecules permit binding interaction of the particle withspecific receptor sites, such as those within the lungs. The particlescan be targeted by attachment of ligands which specifically ornon-specifically bind to particular targets. Exemplary targetingmolecules include antibodies and fragments thereof including thevariable regions, lectins, and hormones or other organic moleculescapable of specific binding for example to receptors on the surfaces ofthe target cells.

[0052] Therapeutic Agents

[0053] Any of a variety of therapeutic, prophylactic or diagnosticagents can be delivered. Examples include synthetic inorganic andorganic compounds, proteins and peptides, polysaccharides and othersugars, lipids, and nucleic acid sequences having therapeutic,prophylactic or diagnostic activities. Nucleic acid sequences includegenes, antisense molecules which bind to complementary DNA to inhibittranscription, and ribozymes. The agents to be incorporated can have avariety of biological activities, such as vasoactive agents, neuroactiveagents, hormones, anticoaguulants, immunomodulating agents, cytotoxicagents, antibiotics, antivirals, antisense, antigens, and antibodies. Insome instances, the proteins may be antibodies or antigens whichotherwise would have to be administered by injection to elicit anappropriate response. Compounds with a wide range of molecular weightcan be encapsulated, for example, between 100 and 500,000 grams permole.

[0054] Proteins are defined as consisting of 100 amino acid residues ormore; peptides are less than 100 amino acid residues. Unless otherwisestated, the term protein refers to both proteins and peptides. Examplesinclude insulin and other hormones. Polysaccharides, such as heparin,can also be administered.

[0055] The porous polymeric aerosols are useful as carriers for avariety of inhalation therapies. They can be used to encapsulate smalland large drugs, release encapsulated drugs over time periods rangingfrom hours to months, and withstand extreme conditions duringaerosolization or following deposition in the lungs that might otherwiseharm the encapsulated therapeutic.

[0056] The porous particles may include a therapeutic agent for localdelivery within the lung, such as agents for the treatment of asthma,emphysema, or cystic fibrosis, or for systemic treatment. For example,genes for the treatment of diseases such as cystic fibrosis can beadministered.

[0057] Administration

[0058] The particles including a therapeutic agent may be administeredalone or in any appropriate pharmaceutical carrier, such as a liquid,for example saline, or a powder, for administration to the respiratorysystem.

[0059] Aerosol dosage, formulations and delivery systems may be selectedfor a particular therapeutic application, as described, for example inGonda, I. “Aerosols for delivery of therapeutic and diagnostic agents tothe respiratory tract,” In Critical Reviews in Therapeutic Drug CarrierSystems, 6:273-313 (1990), and in Moren, “Aerosol dosage forms andformulations,” In Aerosols in Medicine. Principles, Diagnosis andTherapy, Moren, et al., Eds., Esevier, Amsterdam, 1985, the disclosuresof which are incorporated herein by reference.

[0060] The greater efficiency of aerosolization by porous particles ofrelatively large size permits more drug to be delivered than is possiblewith the same mass of nonporous aerosols. The relative large size ofporous aerosols depositing in the deep lungs also minimizes potentialdrug losses caused by particle phagocytosis. The use of porous polymericaerosols as therapeutic carriers provides the benefits of biodegradablepolymers for controlled released in the lungs and long-time local actionor systemic bioavailability. Denaturation of macromolecular drugs can beminimized during aerosolization since macromolecules are contained andprotected within a polymeric shell. Coencapsulation of peptides withpeptidase-inhibitors can minimize peptide enzymatic degradation.

[0061] The present invention will be further understood by reference tothe following non-limiting examples.

EXAMPLE 1

[0062] Synthesis of Porous Poly[(p-carboxyphenoxy)-hexane anhydride](“PCPH”) Particles

[0063] Porous poly[(p-carboxyphenoxy)-hexane anhydride] (“PCPH”)particles were synthesized as follows. 100 mg PCPH (MW-25,000) wasdissolved in 3.0 mL methylene chloride. To this clear solution was added5.0 mL 1% w/v aqueous polyvinyl alcohol (PVA, MW .about.25,000, 88 mole% hydrolyzed) saturated with methylene chloride, and the mixture wasvortexed (Vortex Genie 2, Fisher Scientific) at maximum speed for oneminute. The resulting milky-white emulsion was poured into a beakercontaining 95 mL 1% PVA and homogenized (Silverson Homogenizers) at 6000RPM for one minute using a 0.75 inch tip. After homogenization, themixture was stirred with a magnetic stirring bar and the methylenechloride quickly extracted from the polymer particles by adding 2 mLisopropyl alcohol. The mixture was continued to stir for 35 minutes toallow complete hardening of the microparticles. The hardened particleswere collected by centrifugation and washed several times with doubledistilled water. The particles were freeze dried to obtain afree-flowing powder void of clumps. Yield, 85-90%.

[0064] The mean diameter of this batch was 6.0 μm, however, particleswith mean diameters ranging from a few hundred nanometers to severalmillimeters may be made with only slight modifications. Scanningelectron micrograph photos of a typical barch of PCPH particles showedthe particles to be highly porous. The particles have a mass densityless that 1 g/cm³ as indicated by the fact that the particles float whendispersed in an organic solvent.

EXAMPLE 2

[0065] Synthesis of PLAL-Lys and PLAL-Lys-Ala Polymeric and CopolymericParticles

[0066] Porous PLAL-Lys Particles

[0067] PLAL-Lya particles were prepared by dissolving 50 mg of the graftcopolymer in 0.5 ml dimethylsulfoxide, then adding 1.5 mldichloromethane dropwise. The polymer solution is emulsified in 100 mlof 5% w/v polyvinyl alcohol solution (average molecular weight 25 KDa,88% hydrolyzed) using a homogenizer (Silverson) at a speed ofapproximately 7500 rpm. The resulting dispersion is stirred using amagnetic stirrer for 1 hour. Following this period, the pH is brought to7.0-7.2 by addition of 0.1 N NaOH solution. Stirring is continued for anadditional 2 hours until the methylene chloride is completely evaporatedand the particles hardened. The particles are then isolated bycentrifugation at 4000 rpm (1600 g) for 10 minutes (Sorvall RX-5B). Thesupernatant is discarded and the precipitate washed three times withdistilled water, the dispersion frozen in liquid nitrogen, andlyophilized (Labconco freeze dryer 8) for at least 48 hours. Particlesizing is performed using a Coulter counter. Average particle meandiameters ranged from 100 nm to 14 μm, depending upon processingparameters such as homogenization speed and time. All particles exhibithigh porosity (net mass density less than 0.4 g/cm³). Scanning electronmicrograph photos of the particles showed them to be highly porous.

[0068] Porous PLAL-Ala-Lys Particles

[0069] 100 mg of PLAL-Ala-Lys is completely dissolved in 0.4 mltrifluoroethanol, then 1.0 ml methylene chloride is added dropwise. Thepolymer solution is emulsified in 100 ml of 1% w/v polyvinyl alcoholsolution (average molecular weight 25 KDa, 80% hydrolyzed) using asonicator (Sonic&Materal VC-250) for 15 seconds at an output of 40 W. 2ml of 1% PVA solution is added to the mixture and it is vortexed at thehighest speed for 30 seconds. The mixture is quickly poured into abeaker containing 100 ml 0.3% PVA solution, and stirred for three hoursallowing evaporation of the methylene chloride. Scanning electronmicrograph photos of the particles showed them to be highly porous.

[0070] Porous Copolymer Particles

[0071] Polymeric porous particles consisting of a blend of PLAL-Lys adPLGA-PEG were made. 50 mg of the PLGA-PEG polymer (molecular weight ofPEG: 20 KDa, 1:2 weight ratio of PEG:PLGA, 75:25 lactide:glycolide) wascompletely dissolved in 1 ml dichloromethane. 3 mg ofpoly(lactide-co-lysine)-polylysine graft copolymer is dissolved in 0.1ml dimethylsulfoxide and mixed with the first polymer solution. 0.2 mlTE buffer, pH 7.6, is emulsified in the polymer solution by probesonication (Sonic&Materal VC-250) for 10 seconds at an output of 40 W.To this first emulsion, 2 ml of distilled water is added and mixed usinga vortex mixer at 4000 rpm for 60 seconds. The resulting dispersion isagitated by using a magnetic stirrer for 3 hours until methylenechloride is completely evaporated and microspheres formed. The spheresare then isolated by centrifugation at 5000 rpm for 30 min. Thesupernatant is discarded, the precipitate washed three times withdistilled water and resuspended in 5 ml of water. The dispersion isfrozen in liquid nitrogen and lyophilized for 48 hours.

[0072] Variables which may be manipulated to alter the size distributionof the particles include: polymer concentration, polymer molecularweight, surfactant type (e.g., PVA, PEG, etc.), surfactantconcentration, and mixing intensity. Variables which may be manipulatedto alter the porosity of the particles include: polymer concentration,polymer molecular weight, rate of methylene chloride extraction byisopropyl alcohol (or another miscible solvent), volume of isopropylalcohol added, inclusion of an inner water phase, volume of inner waterphase, inclusion of salts or other highly water-soluble molecules in theinner water phase which leak out of the hardening sphere by osmoticpressure, causing the formation of channels, or pores, in proportion totheir concentration, and surfactant type and concentration.

[0073] By scanning electron microscopy (SEM), the PLAL-Lys-PLGA-PEGparticles were highly porous. The particles had a mean particle diameterof 7 μm±3.8 μm. The blend of PLAL-Lys with poly(lactic acid) (PLA)and/or PLGA-PEG copolymers can be adjusted to adjust particle porosityand size. Additionally, processing parameters such as homogenizationspeed and time can be adjusted. Neither PLAL, PLA nor PLGA-PEG aloneyields a porous structure when prepared by these techniques.

EXAMPLE 3

[0074] Rhodamine Isothiocyanate Labeling of PLAL and PLAL-Lys Particles

[0075] Lysine amine groups on the surface or porous (PLAL-Lys) andnonporous (PLAL) microparticles with similar mean diameters (6-7 μm) andsize distributions (standard deviations 3-4 μm) were labeled withRhodamine isothiaocyanate. The mass density of the porous PLAL-Lysparticles was 0.1 g/cm³ and that of the nonporous PLAL particles was 0.8g/cm³.

[0076] The rhodamine-labeled particles were characterized by confocalmicroscopy. A limited number of lysine finctionalities on the surface ofthe solid particle were able to react with rhodamine isothiocyanate, asevidenced by the flourescent image. In the porous particle, the higherlysine content in the graft copolymer and the porous particle structureresult in a higher level of rhodamine attachment, with rhodamineattachment dispersed throughout the interstices of the porous structure.This also demonstrates that targeting molecules can be attached to theporous particles for interaction with specific receptor sites within thelungs via chemical attachment of appropriate targeting agents to theparticle surface.

EXAMPLE 4

[0077] Aerosolization of PLAL and PLAL-Lys Particles

[0078] To determine whether large porous particles can escape (mouth,throat, and inhaler) deposition and more efficiently enter the airwaysand acini than nonporous particles of similar size, aerosolization anddeposition of porous PLAL-Lys (mean diameter 6.3 μm±3.3 μm) or nonporousPLAL (mean diameter 6.9 μm±3.6 μm) particles were examined in vitrousing a cascade impactor system.

[0079] 20 mg of the porous or nonporous microparticles were placed ingelatin capsules (Eli Lilly), the capsules loaded into a Spinhaler drypowder inhaler (DPI) (Fisons), and the DPI activated. Particles wereaerosolized into a Mark I Andersen Impactor (Anderson Samplers, Ga.)from the DPI for 30 seconds at 28.3 l/min flow rate. Each plate of theAndersen Impactor was previously coated with Tween 80 by immersing theplates in an acetone solution (5% w/vol) and subsequently evaporatingthe acetone in a oven at 60° for 5 min. After aerosolization anddeposition, particles were collected from each stage of the impactorsystem in separate volumetric flasks by rinsing each stage with NaOHsolution (0.2 N) in order to completely degrade the polymers. Afterincubation at 37° C. for 12 h, the fluorescence of each solution wasmeasured (wavelengths of 554 nm excitation, 574 nm emission).

[0080] Particles were determines as nonrespirable (mean aerodynamicdiameter exceeding 4.7 μm: impactor estimate) if they deposited on thefirst three stages of the impactor, and respirable (mean aerodynamicdiameter 4.7 μm or less) if they deposited on subsequent stages. FIG. 1shows that less than 10% of the nonporous (PLAL) particles that exit theDPI are respirable. This is consistent with the large size of themicroparticles and their standard mass density. On the other hand,greater the 55% of the porous (PLAL-Lys) particles are respirable, eventhough the geometrical dimensions of the two particle types are almostidentical. The lower mass density of the porous (PLAL-Lys)microparticles is responsible for this improvement in particlepenetration, as discussed further below.

[0081] The nonporous (PLAL) particles also inefficiently aerosolize fromthe DPI; typically, less than 40% of the nonporous particles exited theSpinhaler DPI for the protocol used. The porous (PLAL-Lys) particlesexhibited much more efficient aerosolization (approximately 80% if theporous microparticles typically exited the DPI during aerosolization).

[0082] The combined effects of efficient aerosolization and highrespirable fraction of aerosolized particle mass means that a fargreater fraction of a porous particle powder is likely to deposit in thelungs than of a nonporous particle powder.

EXAMPLE 5

[0083] In Vivo Aerosolization of PLAL and PLAL-Lys Particles

[0084] The penetration of porous and non-porous polymeric PLAL andPLAL-Lys microparticles into the lungs was evaluated in and in vivoexperiment involving the aerosolization of the microparticles into theairways of live rats.

[0085] Male Sprague Dawley rats (150-200 g) were anesthetized usingketamine (90 mg/kg)/xylazine (10 mg/kg). The anesthetized rat was placedventral side up on a surgical table provided with a temperaturecontrolled pad to maintain physiological temperature. The animal wascannulated about the carina with an endotracheal tube connected to aHarvard ventilator. The animal was force ventilated for 20 minutes and300 ml/min. 50 mg of porous (PLAL-Lys) or nonporous (PLA) microparticleswere introduced into the endotracheal tube.

[0086] Following the period of forced ventilation, the animal waseuthanized and the lungs and trachea were separately washed usingbronchoalveolar lavage. A tracheal cannula was inserted, tied intoplace, and the airways were washed with 10 ml aliquots of HBSS. Thelavage procedure was repeated until a total volume of 30 ml wascollected. The lavage fluid was centrifuged (400 g) and the pelletscollected and resuspended in 2 ml of phenol red-free Hanks balanced saltsolution (Gibco, Grand Island, N.Y.) without Ca²+ and Mg²+ (HBSS). 100ml were removed for particle counting using a hemacytometer. Theremaining solution was mixed with 10 ml of 0.4 N NaOH. After incubationat 37° C. for 12 h, the fluorescence of each solution was measured(wavelengths of 554 nm excitation, 574 nm emission).

[0087]FIG. 2 is a bar graph showing total particle mass deposited in thetrachea and after the carina (lungs) in rat lungs and upper airwaysfollowing intratracheal aerosolization during forced ventilation. ThePLAL-Lys porous particles had a mean diameter 6.9 μm±4.2 μm. Thenonporous particles PLAL particles had a mean diameter of 6.7 μm±3.2 μm.Percent tracheal porous particle deposition was 54.54±0.77, andnonporous deposition was 76.98±1.95. Percent porous particle depositionin the lungs was 46.75±0.77, and nonporous deposition was 23.02±1.95.

[0088] The nonporous (PLAL) particles deposited primarily in the trachea(approximately 79% of all particle mass that entered the trachea). Thisresult is similar to the in vitro performance of the nonporousmicroparticles and is consistent with the relatively large size of thenonporous particles. Approximately 54% if the porous (PLAL-Lys) particlemass deposited in the trachea. Therefore, about half of the porousparticle mass that enters the trachea traverses through the trachea andinto the airways and acini of the rat lungs, demonstrating the effectivepenetration of the porous particles into the lungs.

[0089] Following bronchoalveolar lavage, particles remaining in the ratlungs were obtained by careful dissection of the individual lobes of thelungs. The lobes were placed in separate petri dishes containing 5 ml ofHBSS. Each lobe was teased through 60 mesh screen to dissociate thetissue and was then filtered through cotton gauze to remove tissuedebris and connective tissue. The petri dish and gauze were washed withan additional 15 ml of HBSS to maximize microparticle collection. Eachtissue preparation was centrifuged and resuspended in 2 ml of HBSS andthe number of particles counted in a hemacytometer. The particle numbersremaining in the lungs following the bronchoalveolar lavage are shown inFIG. 3. Lobe numbers correspond to: 1) left lung, 2) anterior, 3)median, 4) posterior, 5) postcaval. A considerably greater number ofporous PLAL-Lys particles enters every lobe of the lungs than thenonporous PLAL particles, even though the geometrical dimensions of thetwo types of particles are essentially the same. These results reflectboth the efficiency of porous particle aerosolization and the propensityof the porous particles to escape deposition prior to the carina orfirst bifurcation.

[0090] Modifications and variations of the present invention will beobvious to those skilled in the art from the foregoing detaileddescription. Such modifications and variations are intended to comewithin the scope of the following claims.

What is claimed is:
 1. An essentially dry composition comprising: spraydried particles for delivering a therapeutic agent to the deep lung,wherein the particles comprise a therapeutic agent and at least 55% ofthe particles have an aerodynamic diameter less than about 4.7 μm asmeasured by a Mark I Anderson Impactor for 30 seconds at 28.3 l/min flowrate.
 2. The composition of claim 1, wherein the particles have a tapdensity less than about 0.4 g/cm³.
 3. The composition of claim 1,wherein the particles have a tap density less than about 0.1 g/cm³. 4.The composition of claim 1, wherein the particles have a mass meandiameter between 5 μm and 30 μm.
 5. The composition of claim 1, whereinthe particles further comprise a pharmaceutically acceptable excipient.6. The composition of claim 5, wherein the pharmaceutically acceptableexcipient is selected from the group consisting of organic compounds,inorganic compounds, surfactants and any combinations thereof.
 7. Thecomposition of claim 6, wherein the pharmaceutically acceptableexcipient is a surfactant.
 8. The composition of claim 1, wherein theagent is selected from the group consisting of proteins, peptides,polysaccharides, lipids, nucleic acids and combinations thereof.
 9. Thecomposition of claim 1, wherein the agent is selected from the groupconsisting of antibodies, antigens, antibiotics, antivirals, hormones,vasoactive agents, neuroactive agents, anticoagulants, immunomodulatingagents, cytotoxic agents, ribozymes, antisense agents and genes.
 10. Thecomposition of claim 1, for use in local inhalation therapy.
 11. Thecomposition of claim 10, wherein the agent is an agent for the treatmentof asthma, emphysema, or cystic fibrosis.
 12. The composition of claim1, for use in systemic inhalation therapy.
 13. The composition of claim12, wherein the agent is insulin.
 14. A method of delivering anessentially dry composition to the deep lung of the pulmonary systemcomprising: administering to the respiratory tract an effective amountof an essentially dry composition comprising spray dried particles fordelivering a therapeutic agent; wherein the particles comprise atherapeutic agent and at least 55% of the particles have an aerodynamicdiameter less than about 4.7 μm as measured by a Mark I AndersonImpactor for 30 seconds at 28.3 l/min flow rate.
 15. The method of claim14, wherein the particles have a tap density less than about 0.4 g/cm³.16. The method of claim 14, wherein the particles have a tap densityless than about 0.1 g/cm³.
 17. The method of claim 14, wherein theparticles have a mass mean diameter between 5 μm and 30 μm.
 18. Themethod of claim 14, wherein the particles further comprise apharmaceutically acceptable excipient.
 19. The method of claim 18,wherein the pharmaceutically acceptable excipient is selected from thegroup consisting of organic compounds, inorganic compounds, surfactantsand any combinations thereof.
 20. The method of claim 19, wherein thepharmaceutically acceptable excipient is a surfactant.
 21. The method ofclaim 14, wherein the agent is selected from the group consisting ofproteins, peptides, polysaccharides, lipids, nucleic acids andcombinations thereof.
 22. The method of claim 14, wherein the agent isselected from the group consisting of antibodies, antigens, antibiotics,antivirals, hormones, vasoactive agents, neuroactive agents,anticoagulants, immunomodulating agents, cytotoxic agents, ribozymes,antisense agents and genes.
 23. The method of claim 14, for use in localinhalation therapy.
 24. The method of claim 23, wherein the agent is anagent for the treatment of asthma, emphysema, or cystic fibrosis. 25.The method of claim 14, for use systemic inhalation therapy.
 26. Themethod of claim 25, wherein the agent is insulin.
 27. The method ofclaim 14, wherein delivery is by dry powder inhaler.